Methods for treatment of multiple sclerosis using peptide analogues at position 91 of human myelin basic protein

ABSTRACT

Peptide analogues of human myelin basic protein containing residues 87-99 are provided. Residue 91 of the peptide analogues is altered from the L-lysine residue found in the native protein to any other amino acid. Pharmaceutical compositions of the peptide analogues are provided. In addition, the peptide analogues are administered to patients with multiple sclerosis.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.08/342,078, filed Nov. 18, 1994, now abandoned.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under contracts NS30201and NS28759, awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

TECHNICAL FIELD

The present invention relates generally to methods for treating andpreventing multiple sclerosis by using peptide analogues of human myelinbasic protein.

BACKGROUND OF THE INVENTION

Multiple sclerosis (MS) is a chronic, inflammatory disease that affectsapproximately 250,000 individuals in the United States. Although theclinical course may be quite variable, the most common form ismanifested by relapsing neurological deficits, in particular, paralysis,sensory deficits, and visual problems.

The inflammatory process occurs primarily within the white matter of thecentral nervous system and is mediated by T lymphocytes, B lymphocytes,and macrophages. These cells are responsible for the demyelination ofaxons. The characteristic lesion in MS is called the plaque due to itsmacroscopic appearance.

Multiple sclerosis is thought to arise from pathogenic T cells thatsomehow evaded mechanisms establishing self-tolerance, and attack normaltissue. T cell reactivity to myelin basic protein may be a criticalcomponent in the development of MS. The pathogenic T cells found inlesions have restricted heterogeneity of antigen receptors (TCR). The Tcells isolated from plaques show rearrangement of a restricted number ofVα and Vβ gene segments. In addition, the TCRs display several dominantamino acid motifs in the third complementarity determining region (CDR),which is the major antigen contact site. All together, three CDR3 motifshave been identified in T cell clones known to recognize an epitopewithin amino acids 86-106 of myelin basic protein. These motifs werefound in 44% of rearranged TCR sequences involving one particular Vβgene rearranged in T cells isolated from brain of two patients with MS.

A definitive treatment for MS has not been established. Historically,corticosteroids and ACTH have been used to treat MS. Basically, thesedrugs reduce the inflammatory response by toxicity to lymphocytes.Recovery may be hastened from acute exacerbations, but these drugs donot prevent future attacks or prevent development of additionaldisabilities or chronic progression of MS (Carter and Rodriguez, MayoClinic Proc. 64:664, 1989; Weiner and Hafler, Ann. Neurol. 23:211,1988). In addition, the substantial side effects of steroid treatmentsmake these drugs undesirable for long-term use.

Other toxic compounds, such as azathioprine, a purine antagonist,cyclophosphamide, and cyclosporine have been used to treat symptoms ofMS. Like corticosteroid treatment, these drugs are beneficial at mostfor a short term and are highly toxic. Side effects include increasedmalignancies, leukopenias, toxic hepatitis, gastrointestinal problems,hypertension, and nephrotoxicity (Mitchell, Cont. Clin. Neurol. 77:231,1993; Weiner and Hafler, supra). Antibody based therapies directedtoward T cells, such as anti-CD4 antibodies, are currently under studyfor treatment of MS. However, these agents may cause deleterious sideeffects by immunocompromising the patient.

More recently, cytokines such as IFN-γ and IFN-β have been administeredin attempts to alleviate the symptoms of MS. However, a pilot studyinvolving IFN-γ was terminated because 7 of 18 patients treated withthis drug experienced a clinical exacerbation within one month afterinitiation of treatment. Moreover, there was an increase in the specificresponse to MBP (Weiner and Hafler, supra).

Betaseron, a modified beta interferon, has recently been approved foruse in MS patients. Although Betaseron treatment showed some improvementin exacerbation rates (Paty et al., Neurology 43:662, 1993), there wasno difference in the rate of clinical deterioration between treated andcontrol groups (IFNB MS Study Group, Neurology 43:655, 1993; Paty etal., supra). Side effects were commonly observed. The most frequent ofsuch side effects were fever (40%-58% of patients), flu-like symptoms(76% of patients), chills (46% of patients), mylagias (41% of patients),and sweating (23% of patients). In addition, injection site reactions(85%), including inflammation, pain, hypersensitivity and necrosis, werecommon (IFNB MS Study Group, supra; Connelly, Annals of Pharm. 28:610,1994).

In view of the problems associated with existing treatments of MS, thereis a compelling need for improved treatments which are more effectiveand are not associated with such disadvantages. The present inventionexploits the use of peptide analogues which antagonize a T cell responseto human myelin basic protein to effectively treat MS, while providingother related advantages.

SUMMARY OF THE INVENTION

The present invention generally provides analogues of human myelin basicprotein, in which the native L-lysine residue at position 91 is altered.Within one aspect of the invention, the analogue is a peptide derivedfrom residues 87-99 of human myelin basic protein (MBP), wherein theL-lysine residue normally found at position 91 of native peptide isaltered to another amino acid. The L-lysine residue at position 91 maybe altered to any other amino acid, and preferably to alanine, serine,glycine, glutamic acid, phenylalanine, arginine, asparagine, histidine,leucine or D-lysine. The alteration is preferably a non-conservativechange or any D-amino acid. The alteration is also preferably one whichresults in reduced production of TNF-α from MBP-reactive T cells.

The present invention provides a pharmaceutical composition comprising apeptide analogue according to the embodiments set out above, in whichthe analogue is contained in a physiologically acceptable carrier ordiluent.

The present invention also provides methods for treating multiplesclerosis by administering to a patient with MS a therapeuticallyeffective amount of a pharmaceutical composition containing analogue asdescribed herein. As noted above, in one aspect a peptide analoguecomprises amino acid residues 87-99 of human myelin basic protein,wherein the lysine at position 91 is replaced by another amino acid.

These and other aspects will become evident upon reference to thefollowing detailed description and attached drawings. In addition,various references are set forth below which describe in more detailcertain procedures or compositions. Each of these references areincorporated herein by reference in their entirety as if each wereindividually noted for incorporation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts DNA and predicted amino acid sequence for human myelinbasic protein.

FIG. 2 is a graph demonstrating competition for MHC binding between MBP(87-99) and the alanine analogue of residue 91 (91K>A). Thealanine-substituted analogue was tested at concentrations ranging from 0to 200 μM for its ability to inhibit the binding of 10 μM biotin-labeledMBP (87-99). The data are presented as the percentage of inhibition ofmean relative binding. Fifty percent inhibition establishes the IC₅₀value.

FIG. 3 is a graph displaying the proliferative response of the T cellline NBI to position 91-substituted analogues. Ten differentsubstitutions were tested. The proliferative response of NBI in responseto concentrations of analogues ranging from 0 to 150 μM was determined.Proliferation is shown as counts per minute. Standard errors of the meanwere less than ±10%. MBP 87-99; peptides from human myelin basic proteincontaining residues 87 to 99; K, lysine; R, arginine; N, asparagine; H,histidine; L, leucine; S, serine; G, glycine; k, D-lysine; E, glutamicacid; F, phenylalanine; and A, alanine.

FIG. 4 is a graph displaying the proliferative response of MBP-reactivelymph node cells to position 91-substituted analogues. Two differentsubstitutions were tested. The proliferative response of lymph nodecells to 10 μM of MBP (87-99), motilin, A91 or K91 was determined.Proliferation is shown as counts per minute. BKG, no peptide added;87-99, MBP (87-99); motilin, an unrelated peptide; A91, peptide analoguewith alanine at position 91; K91, peptide analogue with D-lysine atposition 91.

FIG. 5 is a graph illustrating the ability of alanine-substitutedanalogues to antagonize T cells. The proliferative response of the Tcell line, L87-99, to 2.2 μM MBP (87-99) in the presence of 0.001-0.01μM of the peptide analogue (91K>A) is shown. Results are shown asstimulation index ±SE.

FIG. 6 is a graph depicting the reversal of EAE by soluble peptidetherapy. Rats were injected with 10⁷ L87-99 cells, a procedure whichinduces EAE by adoptive transfer. Five days later, when clinical diseasewas apparent, rats were randomly distributed into three groups of sixrats each. These groups were injected intraperitoneally with 2 mg/ml ofeither MBP (87-99) (91K>A) (--), or PBS (-□-). EAE was graded daily andis presented as a mean score ±SE.

FIGS. 7A and 7B are a pair of graphs showing the amount of IFN-γ andTNF-α production from draining lymph node cells (DLNC). DLNC werestimulated with MBP (87-99) alone () or with the peptide analogue(91K>A) (▪).

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms thatwill be used hereinafter.

“Human myclin basic protein” (“MBP”) refers to a protein found in thecytoplasm of human oligodendroglial cells. The nucleotide sequence andpredicted amino acid sequence of human MBP are presented in FIG. 1 (SEQ.ID Nos. 1 and 2). Although not depicted in FIG. 1, different molecularforms of human myelin basic protein generated by differential splicingor post-translational modification are also within the scope of thisinvention.

“Peptide analogues” of myelin basic protein are derived from residues87-99 of MBP and contain one difference in amino acid sequence betweenthe analogue and native human myelin basic protein, which is adifference at residue 91. Unless otherwise indicated, a named amino acidrefers to the L-form. An L-amino acid from the native peptide may bealtered to any other one of the 20 L-amino acids commonly found inproteins, any one of the corresponding D-amino acids, rare amino acids,such as 4-hydroxyproline, and hydroxylysine, or a non-protein aminoacid, such as α-alanine and homoserine. Also included with the scope ofthe present invention are amino acids which have been altered bychemical means such as methylation (e.g., α-methylvaline), amidation ofthe C-terminal amino acid by an alkylamine such as ethylamine,ethanolamine, and ethylene diamine, and acylation or methylation of anamino acid side chain function (e.g., acylation of the epsilon aminogroup of lysine).

“Residue 91, ” also called “position 91,” refers to amino acid 91 ofhuman myelin basic protein (see FIG. 1; SEQ. ID No. 2) or the amino acidat the comparative position for a peptide derived from MBP. Thenumbering system used relates to the amino acid position within thenative protein, regardless of the length of the peptide or its positionwithin that peptide.

Peptide Analogues of Myelin Basic Protein

As noted above, the present invention provides peptide analogues ofmyclin basic protein in which the naturally occurring L-lysine atposition 91 is altered to another amino acid. The peptide analogues arederived from residues 87-99 of MBP. Residue 91, which is L-lysine in thenative protein, is the key residue. Within this invention, analogueshave an amino acid other than L-lysine at position 91. As noted above,any amino acid alteration at position 91 is within the scope of thisinvention. Preferred peptide analogues include alteration of L-lysine toany one of the following amino acids: D-lysine, alanine, glycine,glutamic acid, phenylalanine, arginine, asparagine, histidine, leucineor serine. These amino acids include both conservative (similar charge,polarity, hydrophobicity, and bulkiness) and non-conservative aminoacids. Although typically one might expect that only non-conservativeamino acid alterations would provide a therapeutic effect, unexpectedlyeven conservative changes (e.g., arginine) greatly affect the functionof the peptide analogue as compared to the native peptide. Suchdiversity of substitution is further illustrated by the fact that thepreferred amino acids noted above are hydrophobic and hydrophilic,charged and uncharged, polar and non-polar.

Peptide analogues may be synthesized by standard chemistry techniques,including synthesis by automated procedure. In general, peptideanalogues are prepared by solid-phase peptide synthesis methodologywhich involves coupling each protected amino acid residue to a resinsupport, preferably a 4-methyl-benzhydrylamine resin, by activation withdicyclohexylcarbodimide to yield a peptide with a C-terminal amide.Alternatively, a chloromethyl resin (Merrifield resin) may be used toyield a peptide with a free carboxylic acid at the C-terminus.Side-chain functional groups are protected as follows: benzyl forserine, threonine, glutamic acid, and aspartic acid; tosyl for histidineand arginine; 2-chlorobenzyloxycarbonyl for lysine and2,6-dichlorobenzyl for tyrosine. Following coupling, thet-butyloxycarbonyl protecting group on the alpha amino function of theadded amino acid is removed by treatment with trifluoroacetic acidfollowed by neutralization with di-isopropyl-ethylamine. The nextprotected residue is then coupled onto the free amino group, propagatingthe peptide chain. After the last residue has been attached, theprotected peptide-resin is treated with hydrogen fluoride to cleave thepeptide from the resin, as well as deprotect the side chain functionalgroups. Crude product can be further purified by gel filtration, HPLC,partition chromatography, or ion-exchange chromatography.

Peptide analogues within the present invention should (a) compete forthe binding of MBP (87-99) to MHC; (b) not cause proliferation of an MBP(87-99)-reactive T cell line; and (c) inhibit induction of EAE(experimental allergic encephalomyelitis) by MBP (87-99) in rodents.

Thus, candidate peptide analogues may be screened for their ability totreat MS by (1) an assay measuring competitive binding to MHC, (2) anassay measuring a T cell proliferation, and (3) an assay assessinginhibition of EAE induction. Those analogues that inhibit binding of thenative peptides, do not stimulate proliferation of MBP-reactive celllines, and inhibit the development of EAE by native peptide, are usefultherapeutics. Although not essential, a further safety assay may beperformed to demonstrate that the analogue does not itself induce EAE.

Binding of peptides to MHC molecules may be assayed on whole cells.Briefly, Lewis rat spleen cells are cultured for 3 hours to allowadherent cells to stick to polystyrene petri dishes. Non-adherent cellsarc removed. Adherent cells, which contain cells expressing MHC class IImolecules, are collected by scraping the dishes. The binding of peptideanalogues to cells is measured by a fluorescence assay. In this assay,splenic adherent cells are mixed with different concentrations ofpeptide analogues and incubated for 1 hour at 37° in a CO₂ incubator.Following incubation, biotin-labeled MBP (87-99) is added to the culturewells. The cells are incubated for another hour and then washed threetimes in medium. Phycoerythrin-conjugated or fluorescein-conjugatedstreptavidin is added along with a fluorochrome-labeled OX-6 or OX-17monoclonal antibody, which reacts with rat MHC Class II I-A and I-E,respectively. The cells are washed twice before analysis by flowcytometry. Fluorescence intensity is calculated by subtracting thefluorescence value obtained from cells stained withphycoerythrin-streptavidin alone (control staining) from thefluorescence value obtained from biotin-labeled MBP (87-99) plusphycoerythrin-streptavidin (experimental staining). Staining withoutanalogue establishes a 100% value. Percent inhibition is calculated foreach analogue and expressed as IC₅₀ values. A peptide analogue with anIC₅₀ value of less than 100 μM is suitable for further screenings.

Candidate peptide analogues are further tested for their property ofcausing or inhibiting proliferation of T cell lines. Two differentassays may be used as alternatives. The first measures the ability ofthe analogue to cause proliferation of T cells in a direct fashion. Thesecond measures the ability of the peptide analogue to inhibitproliferation of T cells induced by native MBP (87-99) peptide.

In the direct proliferation assay, MBP (87-99) reactive T cell lines maybe used as target cells. T cell lines are established from lymph nodestaken from rats injected with MBP (87-99). Lymph node cells are isolatedand cultured for 5 to 8 days with MBP (87-99) and IL-2 as a source of Tcell growth factors. Viable cells are recovered and a second round ofstimulation is performed with MBP (87-99) and irradiated splenocytes asa source of growth factors. After 5 to 6 passages in this manner, theproliferative potential of the cell lines are determined. MBP-reactivelines are used in the proliferation assay. In this assay, T cell linesare cultured for three days with various concentrations of peptideanalogues and irradiated, autologous splenocytes. After three days,0.5-1.0 μCi of [³H]-thymidine is added for 12-16 hours. Cultures areharvested and incorporated counts determined. Mean CPM and standarderror of the mean are calculated from triplicate cultures.

As an alternative to the use of T cell lines as described above,draining lymph node cells from Lewis rats immunized with MBP (87-99) maybe used. Preferably, this assay is used in combination with theproliferation assay using T cell lines. Briefly, Lewis rats are injectedsubcutaneously with MBP (87-99) peptide in complete Freund's adjuvant.Nine to ten days later, draining lymph node cells are isolated andsingle-cell suspensions are prepared. Lymph node cells are incubatedwith various concentrations of peptide analogues for three days in ahumidified air chamber containing 6.5% CO₂. After incubation, thecultures are pulsed with 1-2 μCi of [³H]-thymidine for 12-18 hours.Cultures are harvested on fiberglass filters and counted in ascintillation counter. Mean CPM and the standard error of the mean arecalculated from data determined in triplicate cultures. Peptideanalogues yielding results that are more than three standard deviationsof the mean response with a comparable concentration of MBP (87-99) areconsidered non-stimulatory. Peptide analogues which do not stimulateproliferation at concentrations of less than or equal to 50 μM aresuitable for further screenings.

The second or alternative assay is a competition assay for T cellproliferation. In this assay, antigen presenting spleen cells are firstirradiated and then incubated with native MBP (87-99) peptide for 2-4hours. These cells are then washed and further cultured with T cellsreactive to MBP (87-99). Various concentrations of candidate peptideanalogues are included in cultures for an additional 3 days. Followingthis incubation period, each culture is pulsed with 1 μCi of[³H]-thymidine for an additional 12-18 hours. Cultures are thenharvested on fiberglass filters and counted as above. Mean CPM andstandard error of the mean are calculated from data determined intriplicate cultures. Peptide analogues which inhibit proliferation toapproximately 25% at a concentration of 50 μM or greater are suitablefor further screening.

Candidate peptides that compete for binding of MBP (87-99) to MHC and donot cause direct proliferation of T cell line or can inhibitproliferation by MBP (87-99), are further tested for their ability toinhibit the induction of EAE by MBP (87-99). Briefly, 500 μg of MBP(87-99) is injected as an emulsion in complete Freund's adjuvantsupplemented with heat killed Mycobacterium tuberculosis (H37Ra). Ratsare injected subcutaneously at the base of the tail with 200 μl of theemulsion. Rats are divided into two groups. Approximately 2 days priorto disease induction (usually 10 days following injection of MBP(87-99)) rats are injected intraperitoneally either with PBS or peptideanalogues in PBS. Animals are monitored for clinical signs on a dailybasis by an observer blind to the treatment protocol. EAE is scored on ascale of 0-3: 0, clinically normal; 1, flaccid tail paralysis; 2, hindlimb paralysis; 3, front and hind limbs affected. Peptide analoguesinjected at 5 mg/kg or less (approximately 1 mg per rat) are consideredto inhibit the development of EAE if there is a 50% reduction in themean cumulative score over seven days following onset of diseasesymptoms in the control group.

In addition, as a safety measure, but not essential to this invention,suitable peptide analogues may be tested for direct induction of EAE. Asdescribed in detail in Example 2, various amounts of peptide analoguesare injected at the base of the tail of rats, and the rats examineddaily for signs of EAE. A peptide analogue which is not considered tocause EAE has a mean cumulative score of less than or equal to 1 overseven days when 1 mg (5 mg/kg) in complete Freund's adjuvant isinjected.

Treatment and Prevention of Multiple Sclerosis

As noted above, the present invention provides methods for treating andpreventing multiple sclerosis by administering to the patient atherapeutically effective amount of a peptide analogue of human myelinbasic protein as described herein. Patients suitable for such treatmentmay be identified by criteria establishing a diagnosis of clinicallydefinite MS as defined by the workshop on the diagnosis of MS (Poser etal., Ann. Neurol. 13:227, 1983). Briefly, an individual with clinicallydefinite MS has had two attacks and clinical evidence of either twolesions or clinical evidence of one lesion and paraclinical evidence ofanother, separate lesion. Definite MS may also be diagnosed by evidenceof two attacks and oligoclonal bands of IgG in cerebrospinal fluid or bycombination of an attack, clinical evidence of two lesions andoligoclonal band of IgG in cerebrospinal fluid. Slightly lower criteriaare used for a diagnosis of clinically probable MS.

Effective treatment of multiple sclerosis may be examined in severaldifferent ways. Satisfying any of the following criteria evidenceseffective treatment. Three main criteria are used: EDSS (extendeddisability status scale), appearance of exacerbations or MRI (magneticresonance imaging).

The EDSS is a means to grade clinical impairment due to MS (Kurtzke,Neurology 33:1444, 1983). Eight functional systems are evaluated for thetype and severity of neurologic impairment. Briefly, prior to treatment,patients are evaluated for impairment in the following systems:pyramidal, cerebella, brainstem, sensory, bowel and bladder, visual,cerebral, and other. Follow-ups are conducted at defined intervals. Thescale ranges from 0 (normal) to 10 (death due to MS). A decrease of onefull step defines an effective treatment in the context of the presentinvention (Kurtzke, Ann.Neurol. 36:573-79,1994).

Exacerbations are defined as the appearance of a new symptom that isattributable to MS and accompanied by an appropriate new neurologicabnormality (IFNB MS Study Group, supra). In addition, the exacerbationmust last at least 24 hours and be preceded by stability or improvementfor at least 30 days. Briefly, patients are given a standardneurological examination by clinicians. Exacerbations are either mild,moderate, or severe according to changes in a Neurological Rating Scale(Sipe et al., Neurology 34:1368, 1984). An annual exacerbation rate andproportion of exacerbation-free patients are determined. Therapy isdeemed to be effective if there is a statistically significantdifference in the rate or proportion of exacerbation-free patientsbetween the treated group and the placebo group for either of thesemeasurements. In addition, time to first exacerbation and exacerbationduration and severity may also be measured. A measure of effectivenessas therapy in this regard is a statistically significant difference inthe time to first exacerbation or duration and severity in the treatedgroup compared to control group.

MRI can be used to measure active lesions using gadolinium-DTPA-enhancedimaging (McDonald et al. Ann. Neurol. 36:14, 1994) or the location andextent of lesions using T₂-weighted techniques. Briefly, baseline MRIsare obtained. The same imaging plane and patient position are used foreach subsequent study. Positioning and imaging sequences are chosen tomaximize lesion detection and facilitate lesion tracing. The samepositioning and imaging sequences are used on subsequent studies. Thepresence, location and extent of MS lesions are determined byradiologists. Areas of lesions are outlined and summed slice by slicefor total lesion area. Three analyses may be done: evidence of newlesions, rate of appearance of active lesions, percentage change inlesion area (Paty et al., Neurology 43:665, 1993). Improvement due totherapy is established when there is a statistically significantimprovement in an individual patient compared to baseline or in atreated group versus a placebo group.

Candidate patients for prevention may be identified by the presence ofgenetic factors. For example, a majority of MS patients have HLA-typeDR2a and DR2b. The MS patients having genetic dispositions to MS who aresuitable for treatment fall within two groups. First are patients withearly disease of the relapsing remitting type. Entry criteria wouldinclude disease duration of more than one year, EDSS score of 1.0 to3.5, exacerbation rate of more than 0.5 per year, and free of clinicalexacerbations for 2 months prior to study. The second group wouldinclude people with disease progression greater than 1.0 EDSS unit/yearover the past two years.

Efficacy of the peptide analogue in the context of prevention is judgedbased on the following criteria: frequency of MBP reactive T cellsdetermined by limiting dilution, proliferation response of MBP reactiveT cell lines and clones, cytokine profiles of T cell lines and clones toMBP established from patients. Efficacy is established by decrease infrequency of reactive cells, a reduction in thymidine incorporation withaltered peptide compared to native, and a reduction in TNF and IFN-α.Clinical measurements include the relapse rate in one and two yearintervals, and a change in EDSS, including time to progression frombaseline of 1.0 unit on the EDSS which persists for six months. On aKaplan-Meier curve, a delay in sustained progression of disability showsefficacy. Other criteria include a change in area and volume of T2images on MRI, and the number and volume of lesions determined bygadolinium enhanced images.

Peptide analogues of the present invention may be administered eitheralone, or as a pharmaceutical composition. Briefly, pharmaceuticalcompositions of the present invention may comprise one or more of thepeptide analogues described herein, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like, carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol, proteins,polypeptides or amino acids such as glycine, antioxidants, chelatingagents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide)and preservatives. In addition, pharmaceutical compositions of thepresent invention may also contain one or more additional activeingredients, such as, for example, cytokines like β-interferon.

Compositions of the present invention may be formulated for the mannerof administration indicated, including for example, for oral, nasal,venous, intracranial, intraperitoneal, subcutaneous, or intramuscularadministration. Within other embodiments of the invention, thecompositions described herein may be administered as part of a sustainedrelease implant. Within yet other embodiments, compositions of thepresent invention may be formulized as a lyophilizate, utilizingappropriate excipients which provide stability as a lyophilizate, andsubsequent to rehydration.

Pharmaceutical compositions of the present invention may be administeredin a maimer appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease. Within particularly preferred embodiments of theinvention, the peptide analogue or pharmaceutical compositions describedherein may be administered at a dosage ranging from 5 to 50 mg/kg,although appropriate dosages may be determined by clinical trials.Dosages of peptide analogue will be approximately 5-50 mg/kg, but aredetermined more accurately following trials. Patients may be monitoredfor therapeutic effectiveness by MRI, EDSS, and signs of clinicalexacerbation, as described above.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1 Peptide Synthesis

The peptides were synthesized by solid phase methodology on a peptidesynthesizer (Beckman model 990). Peptides with an amidatedcarboxyl-terminus were prepared with a p-methylbenzhydrylamine resin(MBHA resin); for peptides with a free carboxyl-terminus, a Merrifieldresin coupled with the appropriately protected amino acid was used. Bothresins were obtained from Bachem Fine Chemicals (Torrance, Calif.).Derivatized amino acids (Bachem Fine Chemicals) used in the synthesiswere of the L-configuration unless specified otherwise, and theN-alpha-amino function protected exclusively with the t-butyloxycarbonylgroup. Side-chain functional groups were protected as follows: benzylfor serine, threonine, glutamic acid, and aspartic acid; tosyl forhistidine and arginine; 2-chlorobenzyloxycarbonyl for lysine and2,6-dichlorobenzyl for tyrosine. Coupling of the carboxyl-terminal aminoacid to the MBHA resin was carried out with dicyclohexylcarbodiimide andthe subsequent amino acids were coupled with dicyclohexylcarbodiimideaccording to Ling et al. (Proc. Natl. Acad. Sci. USA 81:4302, 1984).After the last amino acid was incorporated, the t-butyloxycarbonylprotecting group was removed and the peptide-resin conjugate treatedwith a mixture of 14 ml hydrofluoric acid (HF), 1.4 ml anisole, and 0.28ml methylethyl sulfide per gram of resin conjugate at −20° C. for 0.5 hrand at 0° C. for 0.5 hr. HF was removed in vacuum at 0° C., and theresulting peptide and resin mixture was washed twice with diethyl etherand twice with chloroform and diethyl ether alternately. The peptide wasextracted five times with 2 M acetic acid, and the extract lyophilized.The lyophilized product was first purified on a column of Sephadex G-25fine (Pharmacia-LKB, Piscataway, N.J.) developed in 30% acetic acid toremove the truncated fragments and inorganic salts (Ling et al., 1984).Next, peptides were further purified by CM-32 carboxymethylcellulosecation-exchange chromatography (Ling et al., 1984). Final purificationwas achieved by partition chromatography on Sephadex G-25 fine (Ling etal., 1984). The synthetic product was characterized by amino acidanalysis, mass spectrometric analysis, and reversed-phase HPLC.

EXAMPLE 2 Immunizations and EAE Induction

MBP peptides and analogues were dissolved in phosphate-buffered saline(PBS) and emulsified with an equal volume of incomplete Freund'sadjuvant supplemented with 4 mg/ml heat-killed Mycobacteriumtuberculosis H37Ra in oil (Difco Laboratories, Inc., Detroit, Mich.).Rats were immunized subcutaneously in the hind foot pads with 0.1 ml ofthe emulsion and were monitored for clinical signs daily by an observerblind to the treatment protocol. For intravenous injections MBP peptidesand analogues were dissolved in normal saline. EAE was scored asfollows: 0, clinically normal; 1, flaccid tail; 2, hind limb paralysis;3, front and hind limb paralysis.

EXAMPLE 3 Long-term T Cell Lines

Antigen specific long-term T cell lines were derived using the methoddeveloped by Ben-Nun et al. (Eur. J. Immunol. 11:195, 1981). Lewis ratswere injected as described above. Nine to ten days later draining lymphnode cells were cultured (10⁷/ml) for 72 hours in stimulation mediumtogether with 10-20 μM of the injected peptide. The cells were thencollected, washed, and cultured in resting medium. Resting medium wasidentical to the stimulation medium without autologous serum and withthe addition of 10% fetal bovine serum (Gibco) and 12.5% supernatant ofCon A-stimulated splenocytes as a source of T cell growth factors. Con Asupernatant was prepared as described elsewhere (Ben-Nun et al., Eur. J.Immunol. 11:195, 1981). After an additional 5 to 8 days, cells werecollected and either tested for antigen-specific proliferation orcultured for additional cycles.

EXAMPLE 4 MHC Binding Assay

The ability of MBP peptides and peptide analogues to bind MHC wasmeasured. An assay which characterizes the binding of peptides to MHCmolecules on antigen presenting cells (APC) was employed (Mozes et al.,EMBO J. 8:4049, 1989; Gautam et al., PNAS 91:767, 1994). Spleen cellswere cultured in Dulbecco's modified Eagle's medium supplemented with10% fetal bovine serum (Hyclone Laboratories, Logan, Utah) in standardpolystyrene petri dishes (100×15 mm) in a 37° C. incubator containing6.5% CO₂ for 3 hours. Thereafter, non-adherent cells were removed, andthe plates were washed three times with PBS. Adherent cells werecollected using a cell scraper. The binding of MBP (87-99) analogues wasmeasured using a fluorescence assay. Briefly, 5×10⁵ splenic adherentcells in staining buffer (PBS containing 0.1% bovine serum albumin) weremixed with different concentrations ranging from 0-400 μM of MBP (87-99)analogues in individual wells of U-shape 96-well microculture plates andincubated for 1 hr at 37° C. in a 6.5% CO₂ incubator. Followingincubation, 10 μM of biotin-labeled MBP (87-99) was added to culturewells for 1 h. Cells were washed three times with the staining buffer.Phycoerythrin-conjugated or fluoroscein-conjugated streptavidin (BectonDickinson, San Jose, Calif.) was added as a second step reagent (1μg/well) along with 1 μg/well of fluorochrome-labeled OX-6 or OX-17monoclonal antibody (Pharmingen, San Diego, Calif.), which reacts withrat MHC class II I-A or I-E, respectively. The cells were washed twicebefore cytofluorographic analysis on a FACScan (Becton Dickinson).Fluorescence intensity for each sample was calculated by subtracting thefluorescence obtained from OX positive cells stained withphycoerythrin-streptavidin alone (control staining) from thefluorescence obtained from OX positive cells stained with biotin-labeledMBP (87-99) plus phycoerythrin-streptavidin. Percent inhibition wascalculated for each analogue and expressed as IC₅₀ values.

As seen in FIG. 2, the native peptide effectively competed with itselffor binding to APC (IC₅₀=14 μM). The alanine-substitution analogue(91K>A), competed nearly as effectively (IC₅₀=21 μM). These resultsindicate that the amino acid at position 91 can be changed withoutreducing ability of the analogue to be presented to T cells.

EXAMPLE 5 Antigen-specific Lymph Node Cell Proliferation Assay

Female Lewis rats, approximately six weeks old, were purchased fromHarlan Sprague, Indianapolis, Ind. MBP peptides were dissolved inphosphate-buffered saline (PBS) and emulsified with an equal volume ofcomplete Freund's adjuvant (Difco Laboratories, Inc., Detroit, Mich.)supplemented with 4 mg/ml of heat-killed Myobacterium tuberculosis H37Rain oil (Difco). Rats were immunized subcutaneously in the base of thetail with 0.1 ml containing 100 μg of the peptide in the emulsion. Nineto ten days following immunization, rats were sacrificed, their draininglymph node removed and a single cell suspension made. Cells wereresuspended to 5×10⁶ cells per ml in stimulation medium containingDulbecco's modified Eagle's medium (Gibco BRL, Gaithersburg, Md.)supplemented with 2 mercaptoethanol (5×10⁻⁵ M), L-glutamine (2 mM),sodium pyruvate (1 mM), penicillin (100 μg/ml), streptomycin (100μg/ml), and 1% normal rat serum.

For the assay, 100 μl of the lymph node suspension was added to 96-wellflat-bottom wells in the presence of an equal volume of mediumcontaining 10 μM of various peptides (including: motilin as a negativecontrol; MBP87-99; medium only, or alanine or D-amino acid substitutedat position 91). Cultures were then incubated at 37° C. in humidifiedair containing 7.5% CO₂. After 3 days of incubation, 1.0 μCi oftritiated thymidine (20 Ci/mM; New England Nuclear) was added to eachwell and the plates reincubated for an additional 12-16 hours. Theplates were then harvested with a Matrix filtermate harvester (Packard)and counted using an Automatic Direct Beta Counter (Packard). Mean cpmand the standard error of the mean were calculated from triplicatewells.

As seen in FIG. 4, lymph node cells (LNC) reactive to MBP (87-99) wereeffectively stimulated with the immunizing peptide. LNC failed torespond as well to an unrelated peptide, motilin, to media alone or thepeptide analogues (91K>A) and 91K>k).

EXAMPLE 6 Antiaen-specific T Cell Line Proliferation Assays

For this antigen-specific proliferation assay, T cell lines asestablished in Example 3 were used. Cells were plated at a concentrationof 2×10⁴ cells/well with 10⁶ irradiated (2500 rad) splenocytes asaccessory cells together with different concentrations of antigen, andincubated for three days at 37° C. Each well was pulsed with 2 μCi of[³H]-thymidine (specific activity 10 Ci/mmol) for the final 12 to 16hours. Cultures were harvested on fiberglass filters and theproliferative response expressed as CPM±SD or as stimulation index (SI)(mean CPM from test cultures divided by mean CPM from control cultures).

As seen in FIG. 3, the MBP specific rat T cell line responds to thenative peptide; MBP (87-99). An irrelevant peptide motilin (MOT) doesnot stimulate proliferation at any dose. Ten different substitutions ofposition 91 were synthesized and tested in this assay. All ten peptideanalogues failed to stimulate proliferation of the rat T cell line atdoses ranging from 20-120 μM. Thus, for proliferation, no substitutionat position 91 is tolerated.

EXAMPLE 7 Antagonism of T Cell Proliferation Assay

T cell antagonism was detected in a prepulsed proliferation assay asdescribed by De Magistris et al. (Cell 58:625, 1992) with minormodifications. Antigen presenting spleen cells were γ-irradiated (3000rad) and incubated at a concentration of 10⁷ cells/well with 0.2 μM ofthe native peptide in stimulation medium in 10 ml tissue culture platesfor 2.5 hours at 37° C. in a humidified air chamber containing 6.5% CO₂.Spleen cells were then washed and re-cultured at a concentration of5×10⁵ cells/well in U-shape 96-well microculture plates together with5×10⁴ resting anti MBP (87-99) T cell line L87-99. Variousconcentrations of analogues, ranging from 10⁻⁴ μM to 10⁻² μM, were addedfor an additional 60 hours. Each well was pulsed with 1 μCi of[³H]-thymidine (specific activity 10 Ci/mmol) for the final 18 hours.The cultures were then harvested on fiberglass filters and theproliferative response expressed as CPM±SD or as stimulation index (meanCPM from test cultures divided by mean CPM from control cultures). Theanalogue (91K>A) was able to effectively antagonize the response ofL87-99 to native peptide at all concentrations (FIG. 5). Greater than85% inhibition was achieved at 0.01 μM of (91K>A).

EXAMPLE 8 Reversal of EAE

Rats were given 10⁷ L87-99 T cells. All rats developed hind limbparalysis within 5 days. These paralyzed rats were then given a singleinjection (2 mg/ml) of soluble analogue (91K>A) or PBS. All ratsreceiving PBS continued to show hind limb paralysis for the following 4days (FIG. 6, -□-). In contrast, six out of six rats treated withanalogue (91K>A) went into complete remission within 36 hr withoutfurther signs of paralysis (p<0.015) (FIG. 4, --).

EXAMPLE 9 Induction of EAE by Peptide Analogues

The ability of peptide analogues to cause EAE is assessed in vivo. Ratswere injected with MBP (87-99) or (91K>A) peptide analogue as describedin Example 2. Animals were monitored daily for evidence of EAE. Ratsreceiving MBP (87-99) had 100% incidence (18/18 rats) of EAE with a meanmaximum clinical score of 2.4±0.2. In contrast, 0/12 rats receiving thepeptide analogue (91k>A) had EAE. Therefore, this peptide analogue doesnot induce EAE.

EXAMPLE 10 Prevention of EAE by Peptide Analogues

The ability of peptide analogues to prevent EAE when co-injected withEAE-inducing MBP (87-99) peptide was examined. MBP (87-99) was injectedalone or with the peptide analogue (91K>A) in complete Freund's adjuvantat a 1:1 molar ratio. Incidence of EAE, and mean maximum clinical scoredata were collected.

EAE Mean Peptide 1/ Maximum Immunizing Peptides Peptide 2 Clinical GroupPeptide 1 Peptide 2 Ratio Incidence Score 1 MBP(87-99) None 18/18 2.4 ±0.2 2 MBP(87-99) None  0/12 0 [91K>A] 3 MBP(87-99) MBP(87-99) 1:1 6/6 3± 0 4 MBP(87-99) MBP(87-99) 1:1  0/12 0 [91K>A] 5 MBP(68-88) None 6/6 3± 0 6 MBP(68-88) MBP(87-99) 1:1 6/6 3 ± 0 [91K>A]

The table shows that co-immunization of the peptide analogue (91K>A)could specifically inhibit induction of EAE by MBP (87-99), but notinhibit induction of EAE by MBP (68-88), a peptide from a differentregion. Moreover, the peptide analogue did not cause disease.

EXAMPLE 11 TNF-α Production After Treatment With Peptide Analogues

Cytokine production in draining lymph node cells from rats injected withMBP (87-99) alone or with the peptide analogue (91K>A) was determined.IFN-γ and TNF-α production were measured.

Draining lymph node cells (10⁷ cells/ml) were stimulated in vitro withdifferent concentrations of MBP (87-99) or peptide analogue.Supernatants were collected after 24 and 48 hours. IFN-γ was determinedafter 48 hours by use of a rat IFN-γ ELISA kit (GIBCO BRL). TNF-α wasmeasured after 24 hours by ELISA kit (Genzyme Corp., Cambridge, Mass.).

As can be seen in FIG. 7, the peptide analogue caused a marked decreasein cytokine production at all doses greater than 20 μM for TNF-α and atall doses for IFN-γ.

From the foregoing, it will be evident that although specificembodiments of the invention have been described herein for the purposeof illustrating the invention, various modifications may be made withoutdeviating from the spirit and scope of the invention.

2 516 base pairs nucleic acid single linear CDS 1..513 1 ATG GCG TCA CAGAAG AGA CCC TCC CAG AGG CAC GGA TCC AAG TAC CTG 48 Met Ala Ser Gln LysArg Pro Ser Gln Arg His Gly Ser Lys Tyr Leu -1 1 5 10 15 GCC ACA GCA AGTACC ATG GAC CAT GCC AGG CAT GGC TTC CTC CCA AGG 96 Ala Thr Ala Ser ThrMet Asp His Ala Arg His Gly Phe Leu Pro Arg 20 25 30 CAC AGA GAC ACG GGCATC CTT GAC TCC ATC GGG CGC TTC TTT GGC GGT 144 His Arg Asp Thr Gly IleLeu Asp Ser Ile Gly Arg Phe Phe Gly Gly 35 40 45 GAC AGG GGT GCG CCA AAGCGG GGC TCT GGC AAG GAC TCA CAC CAC CCG 192 Asp Arg Gly Ala Pro Lys ArgGly Ser Gly Lys Asp Ser His His Pro 50 55 60 GCA AGA ACT GCT CAC TAT GGCTCC CTG CCC CAG AAG TCA CAC GGC CGG 240 Ala Arg Thr Ala His Tyr Gly SerLeu Pro Gln Lys Ser His Gly Arg 65 70 75 ACC CAA GAT GAA AAC CCC GTA GTCCAC TTC TTC AAG AAC ATT GTG ACG 288 Thr Gln Asp Glu Asn Pro Val Val HisPhe Phe Lys Asn Ile Val Thr 80 85 90 95 CCT CGC ACA CCA CCC CCG TCG CAGGGA AAG GGG AGA GGA CTG TCC CTG 336 Pro Arg Thr Pro Pro Pro Ser Gln GlyLys Gly Arg Gly Leu Ser Leu 100 105 110 AGC AGA TTT AGC TGG GGG GCC GAAGGC CAG AGA CCA GGA TTT GGC TAC 384 Ser Arg Phe Ser Trp Gly Ala Glu GlyGln Arg Pro Gly Phe Gly Tyr 115 120 125 GGA GGC AGA GCG TCC GAC TAT AAATCG GCT CAC AAG GGA TTC AAG GGA 432 Gly Gly Arg Ala Ser Asp Tyr Lys SerAla His Lys Gly Phe Lys Gly 130 135 140 GTC GAT GCC CAG GGC ACG CTT TCCAAA ATT TTT AAG CTG GGA GGA AGA 480 Val Asp Ala Gln Gly Thr Leu Ser LysIle Phe Lys Leu Gly Gly Arg 145 150 155 GAT AGT CGC TCT GGA TCA CCC ATGGCT AGA CGC TGA 516 Asp Ser Arg Ser Gly Ser Pro Met Ala Arg Arg 160 165170 171 amino acids amino acid linear protein 2 Met Ala Ser Gln Lys ArgPro Ser Gln Arg His Gly Ser Lys Tyr Leu -1 1 5 10 15 Ala Thr Ala Ser ThrMet Asp His Ala Arg His Gly Phe Leu Pro Arg 20 25 30 His Arg Asp Thr GlyIle Leu Asp Ser Ile Gly Arg Phe Phe Gly Gly 35 40 45 Asp Arg Gly Ala ProLys Arg Gly Ser Gly Lys Asp Ser His His Pro 50 55 60 Ala Arg Thr Ala HisTyr Gly Ser Leu Pro Gln Lys Ser His Gly Arg 65 70 75 Thr Gln Asp Glu AsnPro Val Val His Phe Phe Lys Asn Ile Val Thr 80 85 90 95 Pro Arg Thr ProPro Pro Ser Gln Gly Lys Gly Arg Gly Leu Ser Leu 100 105 110 Ser Arg PheSer Trp Gly Ala Glu Gly Gln Arg Pro Gly Phe Gly Tyr 115 120 125 Gly GlyArg Ala Ser Asp Tyr Lys Ser Ala His Lys Gly Phe Lys Gly 130 135 140 ValAsp Ala Gln Gly Thr Leu Ser Lys Ile Phe Lys Leu Gly Gly Arg 145 150 155Asp Ser Arg Ser Gly Ser Pro Met Ala Arg Arg 160 165 170

What is claimed is:
 1. A peptide analogue comprising amino acid residues87-99 of human myelin basic protein, wherein lysine at position 91 isaltered to another amino acid selected from the group consisting of aD-amino acid, glycine, glutamic acid, phenylalanine, arginine,asparagine, histidine, leucine, and serine.
 2. The peptide analogue ofclaim 1 wherein the amino acid at position 91 is altered to a D-aminoacid.
 3. The peptide analogue of claim 2 wherein the amino acid atposition 91 is altered to D-lysine.
 4. The peptide analogue of claim 1wherein the alteration at position 91 results in reduced expression ofTNF-α from MBP-reactive T cells upon contacting the MBP-reactive T cellswith the peptide analogue.
 5. A pharmaceutical composition comprising apeptide analogue of amino acid residues 87-99 of human myelin basicprotein, wherein the amino acid at position 91 is altered to D-lysine.6. A method of treating multiple sclerosis, comprising administering toa patient a therapeutically effective amount of a pharmaceuticalcomposition comprising a peptide analogue comprising amino acid residues87-99 of myelin basic protein, wherein the L-lysine residue at position91 is replaced by another amino acid, in combination with aphysiologically acceptable carrier or diluent and wherein said peptideanalogue is not administered in the form of a noncovalent complex with aMajor Histocompatibility Complex (MHC) component.
 7. The method of claim6, wherein the amino acid at position 91 is altered with an amino acidselected from the group consisting of D-lysine, alanine, glycine,glutamic acid, phenylalanine, arginine, asparagine, histidine, leucineand serine.
 8. The method of claim 6, wherein the amino acid at position91 is altered to a non-conservative amino acid.
 9. The method of claim6, wherein the amino acid at position 91 is altered to D-lysine.
 10. Themethod of claim 6, wherein the alteration at position 91 results inreduced expression of TNF-α from MBP-reactive T cells upon contactingthe MBP-reactive T cells with the peptide analogue.